Method for the modulation of function of transcription factors

ABSTRACT

There is provided a method of modulating the function of a transcription factor by administering an effective amount of an oligonucleotide containing optimal nucleotide binding site for the transcription factor. A therapeutic agent having an effective amount of an oligonucleotide for modulating function of a transcription factor and a pharmaceutically acceptable carrier is also provided. Also provided is a treatment of patients having illnesses in which the activation of transcription factors plays a role by administering to a patient an effective amount of an oligonucleotide which competitively binds the related transcription factor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/744,875, filed Apr. 30, 2001, now U.S. Pat. No. 7,002,003, which is aU.S. national stage application of international patent application No.PCT/US99/17366, filed Jul. 30, 1999, which claims the benefit of U.S.Provisional application Ser. No. 60/094,695, filed Jul. 30, 1998.

GOVERNMENT SUPPORT

This invention was made with government support under NationalInstitutes of Health grant number NCI 5 RO1 CA56072. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to transcription factors, which induce thetranscription of genes. More specifically, the present invention relatesto a method of inhibiting transcription factors for use in thetherapeutic treatment of disease associated with hyper-activated signaltransduction pathways, particularly malignancy.

2. Description of Related Art

Transcription factors bind to specific DNA sequences, usually upstreamfrom the coding region of a gene and affect the transcription of thatparticular gene. Signal transducers and activators of transcription(STAT) proteins comprise one family of transcription factors. Both forlaboratory studies and for potential clinical use, interference with thefunction of specific transcription factors is a potentially powerful andhighly selective way of inhibiting the activation of specific genes andof blocking cellular responses (e.g., cell proliferation, apoptosis,differentiation, activation).

The use of oligonucleotides as therapeutic agents may be able tointeract specifically with individual or small numbers of targetmolecules to inhibit expression of disease-causing genes. For a reviewsee Gewirtz et al (1998 Blood 92:712-736). Most of the attention hasbeen placed on antisense oligonucleotides, DNA triplex-formingoligonucleotides, and ribozymes that are engineered to bind to specificDNA or RNA sequences and inhibit transcription or translation of thetarget gene. Generally examples of methods for inhibiting transcriptionfactors that have been proposed include the following.

Small molecule pharmacologic agents might be utilized which interruptsignaling or metabolic pathways thus leading to activation oftranscription factors. These agents have the disadvantage ofnon-specifically inhibiting other cellular molecules and functions andhaving non-specific toxic effects on cells.

Also, anti-sense oligonucleotides can be used. These molecules oftenhave non-specific effects on the target DNA or RNA which are unrelatedto their intended anti-sense inhibition of translation of mRNA.Significant inhibitory effects often are seen even with the controlsense oligonucleotides.

A third method involves dominant-negative mutant transfections. Thisincludes the transfection of cDNA encoding non-functional mutants ofspecific transcription factors or proteins that interact with thetranscription factors, such mutants are non-functional, and alsointerfere with the function of the normal endogenous transcriptionfactor within the cells. These have the disadvantage of the technicaldifficulty of performing the transfections, isolating the cells thatactually are expressing the dominant-negative protein, and regulatingthe level of expression of the dominant-negative protein in the cells.

It would therefore be useful to have a method for inhibitingtranscription factors that does not have these disadvantages and can beused for both in vitro and in vivo use.

SUMMARY OF THE INVENTION

There is provided a method of modulating the function of a transcriptionfactor by administering an effective amount of an oligonucleotidecontaining optimal nucleotide binding sites for the transcriptionfactor. A therapeutic agent having an effective amount of anoligonucleotide for modulating function of transcription factors and apharmaceutically acceptable carrier is also provided. There is providedoligonucleotides having transcription factor modulatory properties. Alsoprovided is a treatment of patients having illnesses in which theactivation of transcription factors play a role, by administering to apatient an effective amount of an oligonucleotide which competitivelybinds the related transcription factor. Also provided is a method ofdetermining prognostic factors associated with particularly malignantcells by determining if particular transcription factors areconstitutively activated.

DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention will be readily appreciated asthe same becomes better understood by reference to the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 shows the sequence of the sense strand of double-stranded DNAfragments that were used for gel mobility assays and for the inhibitionof the activation of STAT5;

FIG. 2 shows the constitutive activation of a STAT-like DNA-bindingfactor in Dami/HEL and Meg-01 cells; Nuclear extracts from each cellline cultured in the absence of cytokine (CTL) or in the presence of asmuch as 400 ng/ml TPO or 40 ng/ml IL-3, were incubated with[³²P]-labeled, double-stranded IRF-1 GAS oligonucleotide; TheDNA-binding complex and unbound probes were separatedelectrophoretically on 5% non-denaturing polyacrylamide gels; Theautoradiograph shows the STAT-like DNA-binding factor (DBF) and thenonspecific bands (NSB);

FIG. 3 shows the identification of the constitutively activatedDNA-binding factor in HEL/Dami and Meg-01 cells by gel electrophoreticmobility supershift assays; Nuclear extracts from HEL/Dami and Meg-01cells without cytokine exposure were incubated with the [³²P]-labeledMGFe probe plus anti-STAT3 antiserum (S3), or with the [³²P]-labeledprobe plus anti-STAT5 antiserum (S5); The autoradiograph shows the STATDNA-binding factor (DBF), nonspecific bands (NSB) and free IRF-1 GASprobe (P); The arrowheads indicate the supershifted complexes, theseresults are representative of three separate experiments; Nuclearextracts also were incubated with the [³²P]-labeled MGFe probe plusanti-STAT1, anti-STAT2 and anti-STAT6 antiserum, no supershiftedcomplexes were observed;

FIG. 4 shows the effects of IRF-1 GAS on the binding of STAT 5 to the[³²P]-labeled MGFe; Nuclear extracts from HEL/Dami and Meg-01 cells wereincubated with the [³²P]-labeled MGFe probe without IRF-1 GAS (NONE) orwith [32P]-labeled probe plus a 100-fold excess of unlabeledoligonucleotide SIE or IRF-1 GAS (I-GAS), respectively; The DNA bindingcomplexes were separated in a 5% non-denaturing polyacrylamide gel; Theautoradiograph shows the STAT transcription factor (STAT5) andnonspecific bands (NSB);

FIG. 5 shows the effects of IRF-1 GAS double-stranded oligonucleotide onHEL/Dami and Meg-01 cell survival and proliferation; HEL/Dami and Meg-01cells were incubated with the indicated concentrations of IRF-1 GASoligo or SIE control oligo with lipid for 72 hours and labeled with 2μCi/ml [³H]-thymidine (TdR); TdR incorporation into newly synthesizedDNA was determined by counting the radioactivity (CPM) from triplicatesamples and expressed as the mean CPM; and

FIG. 6 shows the effects of IRF-1 GAS double-stranded oligonucleotide onHEL/Dami cell survival and proliferation; HEL/Dami cells were incubatedwith the indicated concentrations of STAT1, STAT3, MGFe, STAT5/6 oligoor no oligo control (CTL) with lipid for 72 hours and labeled with 2μCi/ml [³H]-thymidine (TdR); TdR incorporation into newly synthesizedDNA was determined by counting the radioactivity per minute (CPM) fromtriplicate samples and expressed as the mean CPM.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention relates to a method of inhibiting thefunction of transcription factors in viable cells, using specific,double-stranded oligonucleotides that contain the optimal nucleotidebinding site(s) recognized by a given transcription factor. This methodis applicable in vitro for inhibiting DNA binding and function of anytranscription factor that targets a specific nucleotide sequence towhich it binds on native, endogenous DNA to exert its transcriptionalactivation function. The method can also use a specific nucleotidesequence which represents a consensus sequence of the targeted DNA uponwhich the transcription factor acts. Some examples of such transcriptionfactors include (but are not limited to) the STAT family (STATs 1, 2, 3,4, 5a, 5b, and 6), fos/jun, NF kappa B, HIV-TAT, and the E2F family.

The present invention provides a method of inhibiting the function oftranscription factors as shown in the Example herein.

Contrary to that used in the art, i.e. antisense oligonucleotides, DNAtriplex-forming oligonucleotides, and ribozymes that are engineered tobind to specific DNA or RNA sequences, the present invention usesdouble-stranded oligonucleotides that contain, for example, theconsensus STAT5 binding site, TTCNNNGAA, to competitively inhibit theability of activated STAT to bind to its endogenous DNA targets.

As shown in the Examples, the applicants have discovered that growthfactor independent leukemia cells have a constitutively activatedSTAT-like DNA-binding factor (DBF). The STAT-like DBF was found to beSTAT5. It was also discovered that constitutive activation of STAT5correlates with cell proliferation. Furthermore the applicants foundthat cell proliferation could be inhibited by blocking STAT5transcription factor using a double-stranded oligonucleotide containingthe STAT5 binding sequence AGATTTCTAGGAATTCAAATC (SEQ ID NO:1) orGCCTGATTTCCCCGAAATGACGGCA (SEQ ID NO:2) or GTATTTCCCAGAAAAGGAAC (SEQ IDNO:3), which contain the STAT5 consensus binding site TTCNNNGAA, inwhich “N” means any nucleotide. This oligonucleotide with the STAT5binding sequence penetrates into cells and serves as a competitiveinhibitor that binds to activated STAT5 in the cells. Oligonucleotidebound, activated STAT5 is not available to bind to endogenous DNA atSTAT5 binding sites and therefore can not function i.e. activate thetranscription of various cellular genes. Inhibiting the synthesis ofprotein by this method can have many effects on cells. In the currentinvention the inhibition of STAT5 function in this manner, preventedcell proliferation and led to the death of human leukemic cells invitro. Hence, the data is highly predictive of the present inventionbeing effective against human malignant cells.

Studies have shown that there is constitutive activation oftranscription factors, such as STAT3 and STAT5, in human malignant celllines in vitro and in primary malignant cells obtained from fresh humanneoplasms as diverse as acute myeloid leukemias, chronic myeloidleukemias, head and neck carcinomas and breast carcinomas. Activatedtranscription factors are most critical in continuously dividing cellsand during the process of cell division. This indicates a favorabletherapeutic index, since malignant cells are continuously dividing andwould be most affected while normal cell populations having a lowerproliferative index would be less exposed to the oligonucleotide'sinhibitory effect.

In the case of many transcription factors, activation is not critical tonormal cellular functions, even though it may be critical to continuedmalignant cell survival and proliferation. For example, mice in whichboth the STAT5a and STAT5b genes have been knocked out are viable andsurvive to adulthood, suggesting that STAT5 is not essential. Thus,inhibition of activated STAT5 in an acute leukemia patient has asignificant therapeutic effect, and almost no toxic effect. Anyneoplastic disease in which there is an activated transcription factor,which is critical to maintaining survival and proliferation of themalignant cells is a target for this therapeutic approach.

Additionally, the inflammation and tissue destruction in manyimmunologic and inflammatory disorders are driven primarily bycytokines, such as tumor necrosis factor (TNF)-, which acts largelythrough activation of the transcription factor, NF kappa B. Thus, thesequence-specific, double-stranded oligonucleotide containing theconsensus binding site, GGGGACTTTCCC (SEQ ID NO:4), for example theoligonucleotide having the sequence AGTTGAGGGGACTTTCCCAGGC (SEQ ID NO:5)is effective in interfering with the transcriptional activating functionof NF kappa B and blocks the inflammatory response that causes thetissue destruction in such diverse diseases as rheumatoid arthritis, andacute pancreatitis.

Bacterial sepsis has an extremely high morbidity rate. Most evidenceshows that much of the sepsis syndrome is caused by endotoxin-mediatedstimulation of cytokines, which in turn lead to inflammatory andvascular effects. Again, since most pro-inflammatory cytokines result inactivation of transcription factors such as STATs and NF kappa B, acutetreatment with specific double-stranded oligonucleotides designed tocompetitively inhibit the function of these transcription factors is arapid and effective means of interfering with the cytokine-mediatedevents responsible for the major complications of bacterial sepsis.

Recently, Angiotensin II has been shown to activate the JAK2/STAT5signal transduction pathway in cardiac myocytes. In addition,angiotensin-converting enzyme (ACE) inhibitory drugs have been shown tobe an effective clinical treatment in the immediate post-myocardialinfarction period, showing that angiotensin II inhibition is beneficialin this pathologic condition. Thus, STAT5 activation as a result ofexcessive angiotensin II production contributes to the morbidity andmortality of myocardial infarction. Therefore, a novel therapeuticapproach in the early post-infarction period is the use of aSTAT5-binding, double-stranded oligonucleotide as a short-term inhibitorof STAT5 activation.

Also provided by the present invention are therapeutics andpharmaceutical compositions for use in the treatment of patients havingillnesses in which activation of transcription factors play a role. Thetreatment consists of administering to a patient an effective amount ofan oligonucleotide which competitively binds a transcription factor ofthe related illness. These therapeutics and pharmaceuticals which areutilized contain an effective amount of an oligonucleotide formodulating the function of transcription factors and a pharmaceuticallyacceptable carrier. More specifically, there is provided apharmaceutical composition for inhibiting a transcription factor in acell. The pharmaceutical composition contains therein an effectiveamount of a double stranded oligonucleotide, the oligonucleotide havinga sequence bound by a transcription factor.

The composition and therapeutic of the present invention areadministered and dosed in accordance with good medical practice, takinginto account the clinical condition of the individual patient, the siteand method of administration, scheduling of administration, patient age,sex, body weight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the therapeutic, composition and method of treatment of the presentinvention, the compounds of the present invention can be administered invarious ways. It should be noted that it can be administered as thecompounds or as a pharmaceutically acceptable salt and can beadministered alone or as an active ingredient in combination withpharmaceutically acceptable carriers, diluents, adjuvants and vehicles.The compounds can be administered orally, subcutaneously or parenterallyincluding intravenous, intra-arterial, intramuscular,intra-peritoneally, and intra-nasal administration as well as throughintrathecal and infusion techniques. Implants of the compounds are alsouseful. The patient being treated is a warm-blooded animal and, inparticular, mammals including man. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

The above discussion provides a factual basis for the use oftranscription factor inhibitors. The methods used with and the utilityof the present invention can be shown by the following non-limitingexamples and accompanying figures.

EXAMPLES

General Methods:

General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described are generallyfollowed as in Sambrook et al. Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989). Polymerase chain reaction (PCR) is carried outgenerally as in PCR Protocols: A Guide To Methods And Applications,Academic Press, San Diego, Calif. (1990). Reactions and manipulationsinvolving other nucleic acid techniques, unless stated otherwise, areperformed as generally described in Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, andmethodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202;4,801,531; 5,192,659 and 5,272,057 and incorporated herein by reference.In-situ (In-cell) PCR in combination with Flow Cytometry can be used fordetection of cells containing specific DNA and mRNA sequences (Testoniet al, 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known inthe art and not specifically described is are generally followed as inStites et al. (eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and

Shiigi (eds), Selected Methods in Cellular Immunology, W.H. Freeman andCo., New York (1980).

Nuclease Resistance:

Nuclease resistance for the oligonucleotides of the present invention,where needed, is provided by any method known in the art that does notsubstantially interfere with biological activity of theoligodeoxynucleotides as needed for the method of use and delivery [Iyeret al., 1990; Radhakrishnan, et al., 1990; Eckstein, 1985; Spitzer andEckstein, 1988; Woolf et al., 1990; Shaw et al., 1991] and which do notproduce appreciable toxicity. Modifications that can be made to theoligonucleotides in order to enhance nuclease resistance includemodifying the phosphorous or oxygen heteroatom in the phosphatebackbone, short chain alkyl or cycloalkyl intersugar linkages or shortchain heteroatomic or heterocyclic intersugar linkages. These includepreparing methyl phosphonates, phosphorothioates, phosphorodithioatesand morpholino oligomers. In one embodiment it is provided by havingphosphorothioate bonds linking between the four to six 3′-terminusnucleotide bases. Alternatively, phosphorothioate bonds link all thenucleotide bases. Phosphorothioate oligonucleotides do not normally showsignificant toxicity at concentrations that are effective and exhibitsufficient pharmacodynamic half-lives in animals [Agarwal et al., 1996]and are nuclease resistant. Other modifications known in the art may beused where the biological activity is retained, but the stability tonucleases is substantially increased and toxicity not significantlyincreased.

The present invention also includes all analogues of, or modificationsto, an oligonucleotide of the invention that does not substantiallyaffect the transcription factor binding function of the oligonucleotide.Such substitutions may be selected, for example, in order to increasecellular uptake or for increased nuclease resistance as is known in theart. The term may also refer to oligonucleotides which contain two ormore distinct regions where analogues have been substituted.

The nucleotides can be selected from naturally occurring orsynthetically modified bases. Naturally occurring bases include adenine,guanine, cytosine, thymine and uracil. Modified bases of theoligonucleotides include xanthine, hypoxanthine, 2-aminoadenine,6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halocytosine, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil,8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxylguanine and other substituted guanines, other aza and deaza adenines,other aza and deaza guanines, 5-trifluoromethyl uracil and 5-trifluorocytosine.

In addition, analogues of nucleotides can be prepared wherein thestructure of the nucleotide is fundamentally altered and that are bettersuited as therapeutic or experimental reagents. An example of anucleotide analogue is a peptide nucleic acid (PNA) wherein thedeoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replacedwith a polyamide backbone which is similar to that found in peptides.PNA analogues have been shown to be resistant to degradation by enzymesand to have extended lives in vivo and in vitro. Further, PNAs have beenshown to bind stronger to a complementary DNA sequence than a DNAmolecule. This observation is attributed to the lack of charge repulsionbetween the PNA strand and the DNA strand. Other modifications that canbe made to oligonucleotides include polymer backbones, morpholinopolymer backbones [U.S. Pat. No. 5,034,506], cyclic backbones, oracyclic backbones, sugar mimetics or any other modification includingwhich can improve the pharmacodynamics properties of theoligonucleotide.

The oligonucleotides and ribozymes of the present invention can besynthesized by any method known in the art for ribonucleic ordeoxyribonucleic nucleotides. For example, the oligonucleotides can beprepared using solid-phase synthesis such as in an Applied Biosystems380B DNA synthesizer. Final purity of the oligonucleotides is determinedas is known in the art.

Delivery of Therapeutics (Compound):

The therapeutic compound of the present invention is administered anddosed in accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. ThePharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound of the presentinvention can be administered in various ways. It should be noted thatit can be administered as the compound or as pharmaceutically acceptablesalt and can be administered alone or as an active ingredient incombination with pharmaceutically acceptable carriers, diluents,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. The patient being treated is a warm-blooded animal and, inparticular, mammals including man. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

It is noted that humans are treated generally longer than the mice orother experimental animals exemplified herein which treatment has alength proportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses over aperiod of several days, but single doses are preferred.

The doses may be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compounds in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques which deliver it orally or intravenously and retain thebiological activity are preferred.

In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered will vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably will be from 10 μg/kg to 10 mg/kg per day.

Example 1

Reagents

Recombinant human thrombopoietin (TPO), interleukin (IL)-3, IL-6, andgranulocyte macrophage colony-stimulating factor (GM-CSF) were purchasedfrom PeproTech (Rocky Hill, N.J.). Anti-IL-3 and anti-TPO neutralizingantibodies were purchased from R & D system (Minneapolis, Minn.).Anti-phosphotyrosine antibody-agarose and anti-JAK2 antiserum werepurchased from Upstate Biotechnology (Lake Placid, N.Y.).Phosphotyrosine western blotting kit (chemiluminescence) was purchasedfrom Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Anti-goatIgG and anti-mouse IgG antibodies labeled with fluoresceinisothiocyanate were purchased from Zymed (South San Francisco, Calif.).Anti-STAT1, -STAT2, -STAT3, -STAT4, -STAT5 and -STAT6 antibodies werepurchased from Santa Cruz Biotechnology (Santa Cruz, Calif.).[Methyl-3H]-thymidine ([³H]-TdR; Specific activity 70-86 μCi/mmol) and[³²P]-dATP (specific activity >3000 μCi/mmol) were purchased fromAmersham Life Science (Arlington Heights, Ill.).

Cell Lines

Human megakaryocytic leukemic cell line, HEL/Dami, originally describedby Greenberg et al (1988 Blood 72:1968), was obtained from American TypeCulture Collection (ATCC) and was maintained in Iscove's ModifiedDulbecco's Medium (IMDM; GIBCO-BRL, Grand Island, N.Y.) containing 10%horse serum or sera-free nutridoma HEL. Recently, ATCC and DSMZ (GermanCollection of Microorganisms and Cell Cultures) have determined that allsamples of the HEL/Dami cell line that were available for them toanalyze were genetically and karyotypically identical to the humanerythroleukemia cell line (HEL) described previously (Martin 1982Science 216:1233), which has both erythroid and megakaryocyticcharacteristics (Hong 1996 Blood 87:123), for clarity referred to here ias Dami/HEL. Human megakaryoblastic leukemic cell line, Meg-01,originally described by Ogura et al (1985 Blood 66:1384), was obtainedfrom ATCC and was maintained in RPMI 1640 medium (GIBCO-BRL) with 10%heat-inactivated fetal bovine serum (FBS). Human factor-dependentmegakaryoblastic cell line, Mo7e, originally described by Avanzi et al(1988 Br J Haematol. 69:359), was obtained from Genetics Institute(Boston, Mass.) and was maintained in IMDM medium with 10% FBS, 1%L-glutamine and 5 ng/ml GM-CSF. All the cells were incubated at 37° C.in a fully humidified atmosphere with 5% CO₂ in medium alone or in thepresence of various concentrations of cytokines. In experiments todetect the effects of known cytokines or potential cytokines in Dami/HELor Meg-01 conditioned media, Mo7e cells were prepared by washing threetimes with medium and starved for 18 hours in medium without growthfactors prior to cytokine or conditioned medium treatments, as describedby Dusanter-Fourt et al (1994 EMBO J 13:2583). Dami/HEL cells werecultured in IMDM without serum but with 1× Nutridoma HU (BoehringerMannheim Biochemicals, Indianapolis, Ind.), and Meg-01 cells in RPMI1640 with 1× Nutridoma-HU in experiments in which cytokine productionand specific cytokine effects were being studied.

Effects of Cytokines on Cell Growth

To determine the proliferative response of cells to cytokines, DNAsynthesis was measured by [³H]-TdR incorporation. The assays wereperformed in triplicate, using 4×10⁵ cells for Mo7e and Meg-01, or 2×10⁵cells for Dami/HEL cells. Mo7e cells in IMDM with 10% FBS, and Dami/HELand Meg-01 cells in medium with 1× Nutridoma HU were cultured for 72hours without or with cytokines. Subsequently, the cells were labeledwith 2 μCi/ml of [³H]-TdR for an additional 4 hours. Incorporation of[³H]-TdR into newly synthesized DNA (counts per minute; CPM) wasdetermined by liquid scintillation counting, according to a previouslydescribed protocol (Lui 1992 Cancer Res, 52:3667). For the analysis ofcell ploidy, cytokine-starved Mo7e cells (1×10⁶ cells/ml) were culturedin IMDM medium with 10% FBS plus growth factors. Dami/HEL and Meg-01cells (1×10⁶ /ml) were cultured in medium with 1× Nutridoma-HU plusgrowth factors, at 37° C. for 1 to 10 days. The treated cells wereharvested on day 3, 5, 7 and 10. The DNA content of the cells wasquantitated with a FACScan flow cytometer (Becton Dickson, Rutherford,N.J.), following a published protocol (Taylor 1980 J Histochem Cytochem28:1021. Isolated human lymphocytes were used as a diploid control.

Immunoprecipitation and Western Blotting

Unstimulated or cytokine-stimulated cells were lysed in modified RAPIbuffer with 1% NP-40, and extracts were immunoprecipitated, as described(Dusanter-Fourt 1994). The detergent-soluble proteins were incubatedwith anti-phosphotyrosine agarose for 3 hours at 40° C. with shaking.The immunoprecipitates were washed four times with modified RAPI buffer,lysed in SDS buffer and separated by 7.5% non-reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins thenwere transferred to nitrocellulose membrane and probed sequentially withrabbit anti-JAK2 antibody and anti-rabbit IgG antibody labeled withperoxidase. Phosphorylated JAK2 was visualized with enhancedchemiluminescence techniques according to the manufacturer's recommendedprocedure. For the detection of tyrosine phosphorylation of MAPK andother cellular proteins, cells treated with or without cytokines werelysed in SDS buffer, and 30 μg of protein was loaded in each sample lanefor 7.5% SDS-PAGE. The SDS-PAGE-separated proteins were transferred toPVDF membranes and probed with an anti-phosphotyrosine antibody kit(Boehringer Mannheim, Indianapolis, Ind.) or with the PhosphoPlus MAPKantibody kit (New England Biolabs, Beverly, Mass.).

Preparation of Nuclear Extracts and Gel Mobility Shift Assays

Preparation of nuclear extracts and gel mobility shift assays wereperformed according to methods described previously (Yu 1993 Mol CellBiol 13:2011). Briefly, equal amounts of nuclear proteins (5-10 μg) foreach sample were incubated for 30 minutes at 30° C. with 10,000 dpm of[³²P]-labeled double-strand DNA fragment (IRF-1 GAS), which contains theinterferon-γ activation site (GAS) that binds to interferon regulatoryfactor (IRF-1) (5′-GCCTGATTTCCCCGAAATGACGGCA) SEQ ID NO 2 (GORODETSKY1988 GENE 66:87), which contains an identical sequence to that of Bovinemammary gland factor element (MGFe), TTCCCCGAA SEQ ID NO 6. Forcompetition assays, unlabeled FIRE, (5′-AGCGCCTCCCCGGCCGGGGAG) SEQ ID NO7, interferon-stimulated response element (ISG15 ISRE;5′-GATCGGGAAAGGGAAACCGAAACTGAAGCC) SEQ ID NO 8, and sis-inducibleelement (SIE, 5′-AGCTTCATTTCCCGTAAATCCCTAAGC) SEQ ID NO 9 also were usedas potential DNA-protein binding competitors, by adding 50× molar excessof each unlabeled DNA fragment, along with the [³²P]-labeled IRF-1 GASoligonucleotide probe. Unlabeled MGFe was used as a specific competitorfor STAT5 binding to IRF-1 GAS. For gel mobility supershift assays,nuclear extracts were co-incubated with the indicated specific anti-STATantibodies and the [³²P]-labeled oligonucleotide probes. The DNA-proteincomplexes and unbound probe were separated electrophoretically on 5%native polyacrylamide gels in 0.5× TBE buffer (44.5 mM Tris, pH 8.0, 1mM EDTA and 44.5 mM boric acid) for 3 hours at constant 140 volts. Thegels were fixed and dried, and the DNA-protein complexes were visualizedby autoradiography at −70?C. with Kodak X-OMAT film and a DuPont Cranexlightning-plus intensifying screen.

Reverse Transcription/polymerase Chain Reaction (RT/PCR)

Cellular mRNA was prepared with RNAzol™ (Cinna/Bio Tecx Lab, Houston,Tex.) following the protocol suggested by the manufactures. A primer set(TransPrimers, Calbiochem, Calif.) was used to check rearranged BCR/ABLtranscripts. RT/PCR for normal c-ABL also was performed to check RNAquality. A RNA PCR Core kit (GeneAmp, Perkin Elmer, Branchburg, N.J.)was used to perform RT/PCR following the manufacturer's protocol. A 1.8%agarose gel was used for electrophoretic analysis of the PCR products.RT/PCR was performed at least twice to confirm the findings.

Bioassays of Stimulatory Cytokines

Factor-independent Dami/HEL and Meg-01 cells were examined for thecapacity to produce autocrine cytokines. Mo7e cells, the survival ofwhich depends on the presence of any of several growth factors,including GM-CSF, IL-3, IL-6, IL-9, TPO, steel factor/c-kit ligand andTNF-α, were used as a bioassay system to detect possible stimulatoryfactors that might be produced by Dami/HEL or Meg-01 cells. Conditionedmedium was prepared by collecting supernatants from 3-day cultures ofDami/HEL or Meg-01 cells grown in serum-free conditions. The conditionedmedium was incubated with freshly prepared Mo7e cells for 72 hours andcell proliferation was determined by [³H]-TdR incorporation into DNA,following the procedure described in the prior section. IfMo7e-stimulating activity is detected in the medium, it would imply thatthe factor-independent cell lines may produce and secrete autocrinestimulatory factor(s). If no Mo7e proliferation-stimulating activity ofthe conditioned medium is detected, the involvement of humoral factorsin the factor-independent growth of Dami/HEL and Meg-01 cells is reducedsubstantially.

Human Megakaryocytic Leukemic Cell Lines Dami/HEL and Meg-01 Cells Growin a Factor-independent Manner

To determine effects of cytokines on DNA synthesis of the proliferatingcells, various concentrations of cytokines were incubated with Dami/HEL,Meg-01, and Mo7e cells for 3 days and labeled subsequently with [³H]-TdRfor the final four hours of culture. When 0-200 ng/ml of TPO wasincubated with the cytokine-independent cell lines, Dami/HEL and Meg-01,no significant stimulation of [³H]-TdR incorporation was observed. Thecells also were tested for their response to other cytokines, includingGM-CSF, IL-3, IL-6 and TNF-α, and neither Dami/HEL nor Meg-01 cellproliferation is affected by any of these cytokines. Addition ofanti-IL-3 or anti-TPO neutralizing antibodies did not inhibit [³H]-TdRincorporation or cell proliferation. However, when factor-dependent Mo7ecells were incubated with various concentration of TPO, incorporation of[³H]-TdR was stimulated significantly, in a dose-dependent manner. Atthe concentration of 50 ng/ml, TPO regularly stimulates theincorporation of [³H]-thymidine 4.5-fold over that of control Mo7e cellsat 3 days. IL-3, GM-CSF, IL-6, and TNF-α also stimulated Mo7e cellproliferation.

To investigate effects of cytokines on the maturation of thesemegakaryocytic leukemic cell lines, the cells were stained withpropidium iodide, and cell ploidy was analyzed by a FACscan flowcytometer. In the untreated control Dami/HEL, Meg-01 or Mo7e cells, themajority of cells were diploid. When the cells were incubated with100-400 ng/ml of TPO for 1-10 days, no significant increase of cellploidy was observed in any of these cell lines.

STAT-like DNA-binding Factors are Activated Constitutively In Dami/HELand Meg-01 Cells

Applicants analyzed STAT DNA-binding factor activation by gelelectrophoretic mobility shift assays (EMSA) with a [³²P]-labeledoligonucleotide containing the IRF-1 GAS consensus STAT binding site, asthe probe. DNA-binding protein(s) were detected in the nuclear extractsfrom Dami/HEL and Meg-01 cells in the absence of cytokine exposure (FIG.2). Addition of as much as 400 ng/ml of TPO or 40 ng/ml IL-3 does nothave any further effect on the constitutive DNA-binding protein activityin these growth factor-independent cell lines (FIG. 2). Exposure ofthese cells to GM-CSF, IL-6, EPO or TNF-α also did not result insignificant enhancing or inhibitory effects on the constitutiveDNA-binding factor activity. In contrast, no STAT-like DNA-bindingfactor was detectable in untreated control cells

The Constitutive and Cytokine-activated Stat-like Factor BindsSpecifically to DNA Containing the IRF-1 GAS Sequence

Experiments were performed to identify the specific STAT proteinactivated in these megakaryocytic leukemic cell lines. First, using aset of oligonucleotides to attempt to inhibit competitively the bindingof the STAT-like factor to [³²P]-labeled IRF-1 GAS. When Dami/HEL andMeg-01 nuclear extracts were co-incubated with the labeled MGFe probeplus a 100-fold molar excess of unlabeled SIE or IRF-1 GAS (FIG. 4) orFIRE or ISRE oligonucleotides, which do not contain TTCCCCGAA sequence,no competitive inhibition of the formation of DNA-protein complexes wasobserved. However, a 50-fold molar excess of the unlabeled MGFe, whichcontains the same TTCCCCGAA STAT-binding sequence as IRF-1 GAS,completely abolished the formation of the labeled DNA-protein complexes.Similar results were found with Meg-01.

When the nuclear extracts from TPO-treated Mo7e cells were incubatedwith [³²P]-labeled IRF-1 GAS probe or the [³²P]-labeled probe plus a50-fold molar excess of unlabeled FIRE, ISRE, SIE or MGFeoligonucleotides, the cytokine-induced DNA-binding factor in Mo7e cellshad the same features as the constitutively activated STAT-like factorin Dami/HEL cells. Both of the factors are able to bind to the IRF-1 GASprobe, and their binding activity could be abolished completely byunlabeled MGFe, but not by oligonucleotides that do not contain theTTCNNNGAA sequence.

The Constitutively Activated IRF-1 GAS-binding Factor Activated in theDam/HEL and Meg-01 Cells, and the Cytokine-induced IRF-1 GAS-bindingFactor Identified in Mo7E Cell Lines is STAT5

To identify the specific DNA-binding factor(s), gel mobility supershiftassays with antibodies to specific STAT proteins were used to examinethe type of STAT(s) involved in the DNA-protein complexes. Whenanti-STAT5 antiserum was used, supershift of the DNA-protein complexeswas observed with nuclear extracts from Dami/HEL and Meg-01 cellscultured in medium without cytokines (FIG. 3) or from TPO-treated Mo7ecells, but not in untreated Mo7e cells. However, when anti-STAT3antiserum was used, no supershift of DNA-protein complexes occurred inTPO-treated Mo7e or unstimulated Dami/HEL or Meg-01 cells (FIG. 3).Reactions of the DNA-protein complexes with anti-STAT1, anti-STAT2,anti-STAT4 and anti-STAT6 antibodies also were investigated, and nosupershifted DNA-protein complexes were observed. These results indicatethat the predominant or exclusive constitutively activated (in Dami/HELand Meg-01 cells) or cytokine-induced (in Mo7e cells) STAT DNA-bindingfactor in these megakaryocytic leukemic cell lines is STAT5.

The JAK2/STAT5 signal transduction pathway is activated constitutivelyin the growth factor-independent megakaryocytic cell lines, Dami/HEL andMeg-01. In contrast, the activation of this signaling pathway isstrictly cytokine-induced in the growth factor-dependent megakaryocyticcell line, Mo7e. STAT-related transcription factors are activatedconstitutively in primary cells from acute leukemia patients, and asshown in the present invention the close relationship between cellproliferation and the activation of JAK2/STAT5, constitutive activationof the JAK2/STAT5 pathway is believed to be one of the importantmechanisms of leukemogenesis and of maintaining the leukemic phenotype.The STAT signaling pathway may also contribute to oncogenesis.

The accumulated data shows that aberrant expression or mutationalactivation of cytokine receptors or their downstream signal transductionpathways have an important role in abnormally, constitutively activatedsignal transduction pathways and in factor-independent cell growth.

Specific competitive inhibition of activated STAT5 prevented theconstitutively activated STAT5 from inducing proliferation of thesemalignant, leukemic cell lines and resulted in death of the leukemiccells.

Specific oligodeoxynucleotide sequences are known to be preferentialbinding sites for specific transcription factors. For example, theTTCNNNGAA core sequence of the IRF-1 GAS promoter or the mammary glandfactor element (MGFe) is known to be a binding site that is highlyspecific for STAT5.

Oligonucleotides containing the TTCNNNGAA sequence are known to bind andform complexes with STAT5 in vitro. In fact, this forms the basis forthe electrophoretic mobility shift assay (EMSA) that is performedcommonly to detect activated STAT5 (and more generally to detect otherSTATs and other transcription factors). In this application it has beenshown that the STAT5 binding sequence could compete in cells with theability of activated STAT5 to bind to its endogenous DNA targets.Leukemic cell lines HEL/Dami and Meg-01, were found to survive andproliferate in the absence of any growth factor stimulation, expressconstitutively activated JAK2 and STAT5, but no other known JAK or STATproteins or other commonly known signal transduction molecules ortranscription factors could be found to be constitutively activated inthis cell line.

Subsequently, it was found that inhibition of JAK2 activation with asmall molecular pharmacologic agent known as AG490 or inhibition ofactivated STAT5 by transfection of a dominant negative STAT5 mutantinhibits HEL/Dami or Meg-01 cell proliferation. The double-stranded20-mer oligodeoxynucleotide was designed that contains the known STAT5binding sequence (TTCCCCGAA), and was added to the culture of HEL/Damicells, as well as with control oligonucleotide that does not contain theSTAT5 binding sequence. The specific TTCCCCGAA-containingdouble-stranded oligonucleotide inhibits the survival and proliferationof the HEL/Dami and Meg-01 cell lines, whereas the non-specificoligonucleotide does not inhibit specifically the survival andproliferation of these cells. Thus, specific competitive inhibition ofactivated STAT5 prevented the constitutively activated STAT5 frominducing proliferation of this malignant, leukemic cell line andresulted in death of the leukemic cells.

Example 2

STAT5 Activation is Essential for Growth Factor-independent Survival andProliferation of HEL/Dami and Meg-01 Cell Lines:

AG490 (but not PD098059) blocked the constitutive activation of STAT5 inHEL/Dami and Meg-01 cells, but it had no effect on growth factor-inducedMAPK pathway activation. Moreover, AG490 (but not PD098059) inhibitedthe factor-independent proliferation of HEL/Dami and Meg-01 cells in adose-dependent manner. The constitutive activation of JAK2 and STAT5 inHEL/Dami and Meg-01 cells, the inhibition of HEL/Dami and Meg-01 cellproliferation by AG490, and the lack of constitutive activation of theMAPK pathway in these cell lines show that the JAK2/STAT5 pathway isessential for the factor-independent growth of HEL/Dami and Meg-01cells. By using dominant-negative (DN) STAT5 transfections the effect onfactor-independent proliferation of these cell lines that expressconstitutively activated JAK2 and STAT5 was assessed. In order toregulate time and degree of DN-STAT5 expression, the Invitrogenecdysone-inducible expression vector system was used. This involvesdouble stable transfections with one vector (pVgRXR) containing theecdysone receptor cDNA and a second vector (pIND) containing theconstruct of interest (e.g., DN-STAT5 cDNA) driven by anecdysone/muristerone/ponasterone-inducible promoter, with selection inzeocin and G418. HEL/Dami, Meg-01, and Mo7e cells stably transfectedwith the ecdysone receptor, and HEL/Dami cells that also contain theinducible DN-STAT5 construct in pIND were made. There was a significantmuristerone dose-dependent inhibition of HEL/Dami cell proliferationonly in the cells transfected with the DN-STAT5. When HEL/Dami cellswere not transfected or were transfected with only pVgRXR orpIND/DN-STAT5 alone, there was no inhibition of HEL/Dami cellproliferation after addition of muristerone A. Inhibition of HEL/Damicell survival and proliferation occurred only in cells co-transfectedwith pVgRXR and pIND/DN-STAT5. Ponasterone A used instead ofmuristerone, gave the same results.

Interference with the function of specific STAT molecules bydouble-stranded oligonucleotides containing the STAT nucleotide-bindingdomains inhibits the survival and proliferation of AML cells in vitroand in vivo in immunocompromised mice.

(a) Determine Whether Double-stranded Oligonucleotides Containing STAT5Nucleotide-binding Domains Selectively Inhibit the Growth In Vitro ofPrimary Human AML Cells that Express Constitutively Activated STAT5

Double-stranded oligonucleotides containing the TTCNNNGAA binding siterecognized by STAT5, which can competitively inhibit interaction ofSTAT5 with its target endogenous genes, has at therapeutic potential inthose AML patients with constitutively activated STAT5 in their leukemicblast cells.

Primary AML cells are grown in 10% serum or in serum-free medium withNutridoma HU which supports continued growth of multiple cell types. The22-26mer double-stranded phosphorothioate-modified (to reducedegradation and increase T 1/2) oligonucleotides with 2 repeats of theSTAT5 recognition sequence, TTCNNNGAA, are tested to show their abilityto inhibit AML cell survival and proliferation in vitro. Controlcultures contain no oligonucleotides or oligonucleotides to which STAT5does not bind. The oligonucleotides are added into the medium in whichthe cells are growing and the cells are re-fed daily with approximatelya 10 μM concentration of the competitive inhibitory oligonucleotide.Alternatively the oligonucleotides are introduced at (10 nM to 10 μM) inassociation with a cationic lipid (DOTAP or DOSPER). The same cellgrowth assays described above are used and demonstrates competitiveinhibition of STAT5 binding to promoter elements of endogenous DNAinterferes with the growth of primary human AML cells. Variations of themethod can be used to maximize efficiency of AML cell growth inhibitionwhile minimizing normal hemopoietic cell toxicity. Such methods aredirectly applicable to in vitro purging of leukemic cells (and otheractivated STAT-expressing malignant cells) from cells harvested for bonemarrow and peripheral blood stem/progenitor cell transplantation.

(b) Determine Whether Modified Double-stranded OligonucleotidesContaining STAT5 Nucleotide-binding Domains Selectively Inhibit theGrowth of AML Cells Containing Constitutively Activated STAT5 In Vivo inImmunocompromised NOD/SCID mice.

The constitutively activated STAT5-expressing cells that have beenstudied best in vitro (HEL/Dami and Meg-01) are used to determine thetarget doses of oligonucleotides that inhibit growth of human leukemiccells implanted in vivo in NOD/SCID mice. Optimal parameters are definedand used competitively inhibit STAT5 binding to endogenous DNA toinhibit proliferation and hasten apoptotic death of primary AML cellsimplanted in NOD/SCID mice.

Sublethally irradiated (300 cGy of TBI) NOD/SCID mire are used tosupport growth of human AML and blast phase CML cells in a highproportion of cases.

In dose-seeking studies, subcutaneous oligonucleotide doses of 0, 1, 5,10, 20, and 30 μg/gm of body weight/day are administered to 5 mice perdose level, each having 1-5×106 implanted HEL/Dami cells on one flankand Meg-01 cells on the other flank.

The oligonucleotides are biotinylated, and tumors are excised after themice are sacrificed at the end of 4-6 weeks to assess oligonucleotidepenetration into the leukemic cells. The first dose at which at least 3mice have tumors that are at least 50% smaller at 4-6 weeks than micereceiving no oligonucleotide is used in additional studies. The doseseeking studies use an estimated 5 mice/dose level×6 dose levels×2treatments (no oligo vs. functional oligo) with 3 treatment groups—(1)sham treatment, (2) therapeutic STAT5-binding oligonucleotide, and (3) amutated oligonucleotide that does not bind activated. The primaryendpoint is the comparison of the three groups for differences in meantumor size at 4-6 weeks, as measured by 2 perpendicular diameters of thetumor, using a one-way ANOVA. Untreated tumors average about 2×2 cm and95% of untreated tumors are between 1.5 and 3 cm in their largestdiameter at the end of 4 weeks. This study utilizes HEL/Dami leukemiccells (STAT5 constitutively activated) on one flank and TF-1a leukemiccells (no constitutively activated STATs) on the other flank. Anotherexperiment utilizes implantation of Meg-01 leukemic cells (STAT5constitutively activated) on one flank and U266 myeloma cells (STAT3constitutively activated).

The growth of 12 separate samples of primary AML cells (⁻1−5×10⁶ cellsin 5 mice each) as subcutaneous tumor implants or as circulatingleukemic cells (60 mice total) is evaluated. If >50% of primary AMLsfail to grow sufficiently rapidly as tumors, tail vein injections of theprimary AML cells are done to induce systemic leukemia and the survivalof the mice is assessed, using Kaplan-Meier plots. Examination ofmorphology and CD45 expression of cells in the peripheral blood, bonemarrow, and spleen is done on all such mice. Therapeutic studies aredesigned and have statistical considerations that are the same as thosediscussed above for the cell lines, except that each mouse has only oneprimary acute leukemia cell sample implanted on one flank, and aSTAT5-positive control cell line implanted on the opposite flank.

A very high proportion of AML and blast phase CML cells grow well eitheras subcutaneous tumors or systemically in NOD/SCID mice (better than innude mice). Significantly smaller tumor size and/or significantly longersurvival in mice treated with the STAT5-binding oligonucleotide, ascompared with sham treatment or treatment with the non-STAT5-bindingmutant oligonucleotide, which indicates a selective therapeutic benefitof this treatment modality.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

REFERENCES

-   Agrawal, 1996. Antisense oligonucleotides: towards clinical trials,    TIBTECH, 14:376.-   Eckstein 1985. Nucleoside Phosphorothioates. Ann. Rev. Biochem.    54:367-402.-   Iyer et al. 1990. J. Org. Chem. 55:4693-4699.-   Radhakrishnan et al., 1990. The automated synthesis of    sulfur-containing oligodeoxyribonucleotides using    3H-1,2-Benzodithiol-3-One 1,1 Dioxide as a sulfur-transfer    reagent. J. Org. Chem. 55:4693-4699.-   Shaw et al., 1991. Modified deoxyoligonucleotides stable to    exonuclease degradation in serum. Nucleic Acids Res. 19:747-750.-   Spitzer and Eckstein 1988. Inhibition of deoxynucleases by    phosphorothioate groups in oligodeoxyribonucleotides. Nucleic Acids    Res. 18:11691-11704.-   Woolf et al., 1990. The stability, toxicity and effectiveness of    unmodified and phosphorothioate antisense oligodeoxynucleotides in    Xenopus oocytes and embryos. Nucleic Acids Res. 18:1763-1769.

1. A method for treating a person or animal having a disorder associatedwith the activation of a transcription factor, said method comprisingadministering to said person or animal an effective amount of acomposition comprising a double-stranded oligonucleotide, wherein saidtranscription factor binds to said oligonucleotide and activation ofsaid transcription factor is thereby inhibited, wherein saidoligonucleotide comprises the nucleotide sequence AGATTTCTAGGAATTCAAATC(SEQ ID NO:1).
 2. The method according to claim 1, wherein saidtranscription factor is a member of the STAT family of transcriptionfactors.
 3. The method according to claim 1, wherein said transcriptionfactor is STAT5.
 4. The method according to claim 1, wherein saidtranscription factor is activated.
 5. The method according to claim 4,wherein said transcription factor is constitutively activated.
 6. Themethod according to claim 1, wherein said disorder is a neoplasm.
 7. Themethod according to claim 6, wherein said neoplasm is a leukemia orcarcinoma.
 8. The method according to claim 1, wherein said compositionfurther comprises a pharmaceutically acceptable carrier, diluent, and/oradjuvant.
 9. A method of inhibiting the function of a transcriptionfactor in a cell, said method comprising contacting a cell with adouble-stranded oligonucleotide. wherein said oligonucleotide is takeninto said cell and said transcription factor binds to saidoligonucleotide and function of said transcription factor is therebyinhibited, wherein said oligonucleotide comprises the nucleotidesequence AGATTTCTAGGAATTCAAATC (SEQ ID NO:1).
 10. The method accordingto claim 9, wherein said transcription factor is activated.
 11. Themethod according to claim 10, wherein said transcription factor isconstitutively activated.
 12. The method according to claim 9, whereinsaid cell is a malignant cell.
 13. The method according to claim 9,wherein said cell is a leukemia cell or a carcinoma cell.
 14. The methodaccording to claim 9, wherein said transcription factor is a member ofthe STAT family of transcription factors.
 15. The method according toclaim 9, wherein said transcription factor is STAT5.
 16. The methodaccording claim 1, wherein said oligonucleotide comprises two or morecopies of said nucleotide sequence.
 17. The method according claim 1,wherein said oligonucleotide consists of the nucleotide sequenceAGATTTCTAGGAATTCAAATC (SEQ ID NO:1).
 18. The method according to claim9, wherein said oligonucleotide comprises two or more copies of saidnucleotide sequence.
 19. The method according to claim 9, wherein saidoligonucleotide consists of the nucleotide sequenceAGATTTCTAGGAATTCAAATC (SEQ ID NO:1).
 20. The method according to claim7, wherein said leukemia is acute myeloid leukemia or chronic myeloidleukemia.
 21. The method according to claim 7, wherein said carcinoma isa carcinoma of the head, neck, or breast.
 22. The method according toclaim 1, wherein said composition is administered orally,subcutaneously, or parenterally.
 23. The method according to claim 22,wherein said composition is administered intravenously, intraarterially,intramuscularly, intraperitoneally, or intranasally.
 24. The methodaccording to claim 1, wherein said oligonucleotide is modified toincrease resistance of said oligonucleotide to nucleases.
 25. The methodaccording to claim 9, wherein said oligonucleotide is modified toincrease resistance of said oligonucleotide to nucleases.